Cloning, Purification And Biochemical Characterization Of A Novel TldD/PmbA Family Protease (TPP) From Thermophilic Archaea

Protease is a kind of to hydrolysis of the peptide bond hydrolysis enzyme, widely exists in animals, plants and microorganisms. Most organisms typically employ 2–4% of their genetic resources to code for proteases, which are one of the most abundant classes of enzymes and are involved in a wide range of biological processes, including cell-cycle progression, cell signalling, proliferation and death, protein trafficking and immune response. Proteases are one of the most important groups of industrial enzymes They are one of the three largest groups of industrial enzymes. Proteases have a large variety of applications, mainly in the food processing, feed, metal recovery, detergent, leather processing, waste treatment, and chemical industries, as well as for medical uses. Proteases can hydrolyze the protein in the pathogen cell wall, which have a great potential applications in biological control. Currently used in industry of protease exists many problems, such as thermal stability, p H stability stability, enzyme activity is not ideal and so on. The enzymes with extreme art are used by optimization due to the high physical and chemical stability of the enzymes are necessary. People try to obtain the enzymes from microorganism existing in the extreme environment.T. kodakarensis KOD1 is a kind of thermophilic anaerobic archaea, which exists in the extreme environment. And the enzymes might be have the similar characteristics from T. kodakarensis KOD1. In this stady, the gene Tk0499 encoding a novel Tld D/Pmb A family protease(TPP) from Thermococcus kodakarensis KOD1 was identified and characterized. In order to establish a foundation for further analysis of the structure and characterization of the protease, we did the following works.1. Cloning, purification and sequence analysis of TPP from Thermococcus kodakarensis KOD1. The protease encoding gene Tk0499 was cloned from genome DNA of T. kodakarensis KOD1. The amplified fragment was inserted into p ET28a(+) to construct expression vector which was expressed in induced with isopropyl-β-D-thiogalactopyranoside. The recombinant protease was purified by affinity chromatography and the sequence analysis of TPP was dependented on NCBI-Blast data. The gene encodes a protein of 441 amino acid residues with a molecular mass of 48.5 k Da and the protein belongs to Tld D/Pmb A family protease which is peculiar to eukaryotic.2. Characterization of TPP. We further researched its proteolytic activity biochemically by overexpression of the recombinant TPP in Escherichia coli BL21(DE3) and characterization of the purified enzyme. Firstly, we examined the ability of TPP to proteolyse casein using SDS-PAGE. The result suggests that the purified protease TPP has no obvious proteolytic activity against casein. TPP exhibited self-cleavage activity that required placed in 4℃, and the capacity of self-cleavage activity decreased with raising of the temperature. Mass Spectrometry analysis suggested that TPP finished the self-cleavage phenomenon by cutting it’s I25 and I427 to generate short forms(S-TPP). How ever, S-TPP showed higher efficiency activity of degraded casein than L-TPP uncleaved TPP forms are considered as TPP long form.3. Characterization the active form S-TPP under high temperature. Cloning, purification and characterization of S-TPP using method as used for TPP. We also examined the ability of cloned S-TPP to proteolyse casein using method as used for TPP activity of degraded casein. The SDS–PAGE analysis explains that the cloned S-TPP showed a higher proteolytic activity against casein.4. Assays for S-TPP activity. The cloned S-TPP showed similar activity to S-TPP. S-TPP was active in a broad range of p H conditions(p Hs 4.0–10.0), with an optimal p H of 7.0 and a temperature optimum of 40℃In this report, we cloned and overexpressed a Tld D/Pmb A family protease from the archaeon T. kodakarensis KOD1(TPP). We have biochemically characterized the enzyme and demonstrated its optimal enzymatic condition. The results demonstrated that low temperature dependent autocatalytic cleavage in a putative protease of T. kodakarensis KOD1 makes it an active form under high temperature. Our works establish a foundation for further analysis of the structure of the protease and biochemistry.